Necrotizing enterocolitis (NEC) is a devastating disease that costs the US healthcare system US$5 billion annually. It primarily affects preterm infants and there are no effective targeted strategies for prevention or treatment. The pathogenesis remains poorly understood but prematurity is the most significant risk factor and an altered microbiota and premature immune response are important in the pathogenesis. The risk for prematurity increases in babies born to mothers whom are obese/overweight (50% of childbearing age women in the US), and our maternal high fat diet model (mHFD) results in increased susceptibility to NEC mediated by altered bacterial colonization and innate immunity. Our objective is to utilize this model to unravel how mHFD alters neonatal colonization and immunity to increase susceptibility to NEC. This proposal utilizes a novel murine mHFD model, now established in our lab. We have recently shown with this model that mHFD exposure results a unique post-natal colonization pattern (microbiota) in offspring. This altered microbiota subsequently increases inflammatory type 3 innate lymphoid cells (ILC3), resulting in increased susceptibility to NEC-like injury. We have preliminary data that strongly suggests that exposure to bacteria-dependent ligands in-utero that signal via toll-like receptors (TLR) are responsible for the altered microbiota in mHFD offspring. In addition, introduction of an aryl hydrocarbon receptor (Ahr) ligand-producing bacteria (L. murinus) expands these ILC3 in neonatal offspring. L. murinus is specifically increased in mHFD offspring. We therefore hypothesize that exposure to mHFD results in altered in utero TLR signaling and subsequent post-natal colonization with Ahr-ligand producing bacteria that expand ILC3, resulting in increased susceptibility to intestinal inflammation in offspring. To test this, we have 2 specific aims: (1) Determine how in utero TLR-mediated signaling regulates mHFD alteration of post-natal colonization in offspring and (2) Determine how ligands of mHFD microbiota modulate Ahr signaling to educate ILC3 in offspring. We will test specific aim 1 by utilizing germ-free mice, conditional MyD88 knock-out mice (no TLR signaling via MyD88) and specific TLR deficient mice placed on mHFD. Specific aim 2 will be examined utilizing in vivo and in vitro (sort-purified ILC3 and intestinal explants) introduction of known Ahr ligands and antagonists. Adoptive transfer experiments will also be performed with pre-treated ILC3s in neonatal mice exposed to our NEC model. At the completion of these studies, we will dramatically advance our knowledge of how maternal nutrition affects post-natal colonization in offspring and further identify generalizable and novel in-utero and post-natal approaches to altering the microbiota in neonates and modulating ILC3 function to prevent NEC.